The first discovery of Polypedatesteraiensis (Dubois, 1987) (Rhacophoridae, Anura) in China

Abstract Background The genus of Polypedates Tschudi, 1838 currently comprises 25 recognised species with four of these species reported in Yunnan, China. Dubois (1987) speculated the distribution of P.teraiensis in China; however, there was no study carried out to confirm its distribution in the region. New information We herein describe P.teraiensis as a new national record, based on a specimen collected from Yunnan border region. Phylogenetically, our sequence clustered with the sequences of recognised P.teraiensis specimens from Bangladesh, Myanmar and India. The uncorrected pairwise distances between the specimens from China and other P.teraiensis localities was small, ranging from 0.0-0.7%, based on 16S rRNA gene. Therefore, we report P.teraiensis as a new species record for China.

During our field investigation of amphibians and reptiles of the Gaoligong Mountain in 2023, we collected a single specimen from Yingjiang County in south-western Yunnan Province, China, close to the China-Myanmar-border. Based on phylogenetic analysis, we identified it as conspecific with P. teraiensis, which represents the first confirmed voucher for the speculated distribution of the species in China.Here, we confirm the distribution of P. teraiensis in China and provide a description of the Chinese specimen.

Materials and methods
The field survey was conducted in the Tongbiguan Provincial Nature Reserve under the permit issued by the Dehong Prefecture Forestry and Grassland Bureau of Yunnan Province (Fig. 1).The specimen was photographed, then euthanised and fixed in 75% ethanol for permanent storage.A liver tissue sample was collected and preserved in absolute ethyl alcohol for molecular analysis.The specimen was deposited at the Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences (KIZ).
Morphological measurements were taken using a digital caliper to the nearest 0.1 mm (Suppl.material 2).Morphological terminology followed Fei et al. (2009) Genomic DNA was extracted from the liver tissue using the standard phenol-chloroform extraction protocol (Sambrook et al. 1989).Partial fragments of the mitochondrial 16S rRNA were amplified and sequenced for this sample using the primer pairs (5'-3') 16S rRNA-F (CGCCTGTTTAYCAAAAACAT) and 16S rRNA-R (CCGGTYTGAACTCAGAT CAYGT) (Kocher et al. 1989).The polymerase chain reaction (PCR) was performed in a 25 μl reaction volume with the following cycling conditions: initial denaturation step at 95°C for 5 min, 35 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min and final extension at 72°C for 10 min.The products were purified and sequenced by Tsingke Biotechnology (Beijing) Co., Ltd., using the same primers employed in the PCR process in both forward and reverse directions.Sequencing was performed using the BigDye Terminator Cycle Sequencing Kit on an ABI PRISM 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA).The newly-obtained nucleotide sequence was first assembled and edited using DNASTAR LASERGENE 7.1.after which the sequence was deposited in the GenBank.
Phylogenetic reconstruction was performed using Bayesian Inference (BI) and Maximum Likelihood (ML) methods, based on the 16S rRNA gene.The best-fit substitution model of evolution was selected under the Bayesian Information Criterion (BIC; Posada ( 2008)) by the programme jModelTest 2. 1.7 (Darriba et al. 2012).BI analysis was implemented by the CIPRES web server (Miller et al. 2010).The BI analyses were conducted with 10 million generations using the SYM+G model and sampled every 1000 generations.Convergence was assessed in Tracer 1.5 (Rambaut and Drummond 2009), based on having an average standard deviation of split frequencies less than 0.01 and ESS values great than 200.We excluded the first 25% of trees as burn-in before the log-likelihood scores stabilised.Maximum Likelihood analyses were performed using RAxML-HPC BlackBox 8.2.10 (Stamatakis 2014) on the CIPRES Science Gateway ( Miller et al. 2010).The analyses used the proportion of invariable sites estimated from the data and 1,000 bootstrap pseudoreplicates under the GTR+gamma model.

Data resources
The aligned 16S rRNA dataset contained a total of 507 nucleotide base pairs (bp), with 165 variable positions and 126 parsimony informative sites (including outgroups).The ML and BI trees had essentially identical topologies and most terminal clades obtained relatively high support values, except for some internal nodes (Fig. 2).The male specimen collected from Tongbiguan Township, Yingjiang County, Yunnan, China, clustered with the specimens of P. teraiensis from Myanmar, India and Bangladesh with strong support (Bayesian posterior probability (BPP) = 1.00, bootstrap support (BS) = 98) for both Bayesian Inference and Maximum Likelihood analysis (Fig. 2).The genetic distance (uncorrected p-distance) between the specimen from China and the specimens of P. teraiensis from other regions was found to be very small (0.0-0.7%, Suppl.material 3).According to phylogenetic data, we confirm the specimen from Tongbiguan Nature Reserve as P. teraiensis and we provide a detailed description of the Chinese specimen.to snout than eyes (SN/N-EL 43.4%); internarial distance less than interorbital distance (IND/IOD 79.2%), but larger than upper eyelid width (IND/UEW 105.0%); eyes large, about one-third of head length (ED/HL 32.7%); tympanum distinct, oval, larger than two-third of eye diameter (TD/ED 69.6%); supratympanic fold distinct, slender, extend from posterior eye to above shoulder; maxillary teeth single row, small; tongue heartshaped, deeply notched posteriorly, posterior 1/3 free; vomerine teeth two short rows, prominently, untouching inner front edges of choanae, separated by distance less than length of each series; male with internal subgular vocal sacs, vocal sac opening on floor of mouth at each corner (Fig. 3).
Dorsal skin relatively smooth with small granular, dorsolateral fold absent; throat smooth with small tubercles, indiscernible; chest, belly and ventral thigh speckled with small tubercles (Fig. 3).

Colouration in preservative
Dorsal surface is faded to greyish-brown, mottled with dark patches; transverse bars on the back of the limbs; black stripes under the temporal fold; ventral surface creamy white, with brown pigment particles on the chest.

Distribution
Our study further extends the species' distribution range to Yunnan, China.Therefore, the species currently known from the Dehong Prefecture, south Yunnan, China, eastern Nepal, Bhutan, India, Bangladesh and Myanmar.

Ecology
This species often inhabits evergreen broad-leaved forests.We found it in the shrubbery by the stream during summer nights.This species is in sympatric distribution with Amolops afghanus and Zhangixalus smaragdinus.

Discussion
Yunnan has a rich species diversity and is often referred to as the "kingdom of animals".Within it lies the Gaoligong Mountain situated in the border region between western Yunnan, China and Myanmar, at the intersection of three biodiversity hotspots (the Himalayas, Indo-Burma and the mountains of southwest China) (Myers et al. 2000, Mittermeier et al. 2004).This region boasts a rich species diversity, especially the Yingjiang County located in the southern part of the Gaoligong Mountain (Li et al. 2024, Wu et al. 2024).Over the years, several new species and new species records have been continuously discovered in this region, an indicator that the area's biodiversity may have been greatly underestimated, thus underscoring the need for further exploration and investigation efforts (e.g.Yang and Chan 2018, Yang et al. 2018, Wu et al. 2021, Yu et al. 2022, Zhang et al. 2022, Wu et al. 2023, Wu et al. 2024).Following our field surveys of the Gaoligong Mountain, we recorded three species of Polypedates.Our increased the known species number of Polypedates in China to five, further supporting the conclusion that amphibian diversity in the Gaoligong Mountain still remains underestimated.
Our field survey also revealed that three species of the genus Polypedates are sympatric: P. braueri, P. impresus and P. teraiensis.These three sympatric species from the Tongbiguan Provincial Nature Reserve, Yunnan Province, are difficult to distinguish from each other, solely based on morphological evidence.The application of molecular methods is crucial for reliable identification as it guides morphological re-examinations, further elucidating fine-scale differences in morphological characteristics that represent speciesspecific variations.Moreover, our understanding of the mechanisms driving sympatric speciation in these species remains limited.Future studies that integrate additional data, such as acoustic evidence and genomics, hold promise in addressing this question comprehensively.

Figure 1 .
Figure 1.Map showing the location of the new record of P. teraiensis in China (red circle).

Figure 3 .
Figure 3. Male of Polypedates teraiensis (KIZ 051716) in preservative.A Dorsal view; B Ventral view; C Ventral view of finger; D Ventral view of toe.